Photodynamic therapy (PDT) of cancer and of other conditions continues to gain clinical acceptance. The approvals of three photosensitizing drugs, Photofrin, Visudyne, and Levulan, in the United States and the ongoing clinical evaluation of several promising new agents throughout the world provide the context for ongoing laboratory studies that are designed to understand further and to optimize this therapy. During the past several years, the field has gained a deeper appreciation of the complex and dynamic interactions among the photosensitizing drug, light, and oxygen that together define the dosimetry of PDT. For more than a decade, research in this laboratory has demonstrated the existence and emphasized the consequences of microscopic gradients of oxygen and of photosensitizer in tumors, explored several forms of optical spectroscopy and imaging as means of monitoring photodynamic dose deposition and evaluating biological response to PDT, and more recently, undertaken the use of fluorescent reporters to image PDT-induced gene expression in vivo. Progress during the previous funding cycle has created a number of opportunities to make further significant progress on these and closely related problems. Toward these general ends, the application poses the following three specific aims: (1) to further expand the experimental investigation and theoretical analysis of microscopic heterogeneities in photosensitizer concentration, oxygen availability, and dose deposition;(2) to exploit molecular imaging strategies to determine heterogeneities in tumor response to PDT and to develop means of imaging the mechanisms governing the recruitment of immune responses;and (3) to measure optical properties of human renal cell carcinoma and of normal kidney and to explore means of further establishing image-guided, interstitial PDT for deep tumors. The experimental methods that will be used to accomplish these aims include laser scanning optical sectioning fluorescence microscopy and microspectrofluorimetry, whole mouse fluorescence imaging and spectroscopy in vivo, and diffuse reflectance spectroscopy. Relevance to public health: Photodynamic therapy is approved for the treatment of several cancers and pre-cancerous conditions. Its potential will be more fully realized as we understand more completely the factors that limit its effectiveness and translate findings from the laboratory to the clinic. Studies proposed in this grant address both of these objectives.